Measurement of light energy is becoming a very attractive method for monitoring the presence or concentration of substances in various media. Numerous bioluminescent and chemiluminescent reaction systems have been devised (Schroeder, et al., Methods in Enzymology 17:24-462 (1978); Zeigler, M. M., and T. O. Baldwin, Current Topics In Bioenergetics, D. Rao Sanadi ed., (Academic Press) pp. 65-113 (1981); DeLuca, M., Non-Radiometric Assays: Technology and Application in Polypeptide and Steroid Hormone Detection, (Alan R. Liss, Inc.) pp. 47-60 and 61-77 (1988); DeJong, G. J., and P. J. M. Kwakman, J. of Chromatography 492:319-343 (1989); McCapra, F. et al., J. Biolumin. Chemilumin. 4:51-58 (1989); Diamandis, E. P., Clin. Biochem. 23:437-443 (1990); Gillevet, P. M., Nature 348:657-658 (1990); Kricka, L. J., Amer. Clin Lab., November/December:30-32 (1990)).
Luminescence is the production of light by any means, including photoexcitation or a chemical reaction. Chemiluminescence is the emission of light only by means of a chemical reaction. It can be further defined as the emission of light during the reversion to the ground state of electronically excited products of chemical reactions (Woodhead, J. S. et al., Complementary Immunoassays, W. P. Collins ed., (John Wiley & Sons Ltd.), pp. 181-191 (1988)). Chemiluminescent reactions can be divided into enzyme-mediated and nonenzymatic reactions. It has been known for some time that the luminescent reactant luminol can be oxidized in neutral to alkaline conditions (pH 7.0-10.2) in the presence of oxidoreductase enzymes (horseradish peroxidase, xanthine oxidase, glucose oxidase), H.sub.2 O.sub.2, certain inorganic metal ion catalysts or molecules (iron, manganese, copper, zinc), and chelating agents, and that this oxidation leads to the production of an excited intermediate (3-aminophthalic acid) which emits light on decay to its ground state, (Schroeder, H. R. et al., Anal. Chem. 48:1933-1937 (1976); Simpson, J. S. A. et al., Nature 279:646-647 (1979); Baret, A., U.S. Pat. No. 4,933,276)). Other specific molecules and derivatives used to produce luminescence include cyclic diacyl hydrazides other than luminol (e.g., isoluminols), dioxetane derivatives, acridinium derivatives and peroxyoxylates (Messeri, G. et al., J. Biolum. Chemilum. 4:154-158 (1989); Schaap, A. P. et al., Tetrahedron Lett. 28:935-938 (1987); Givens, R. S. et al. ACS Symposium Series 383; Luminescence Applications, M. C. Goldberg ed., (Amer. Chem. Soc., Wash. D.C., pp. 127-154 (1989)). Additional molecules which produce light and have been utilized in the ultrasensitive measurement of molecules are polycyclic and reduced nitropolycyclic aromatic hydrocarbons, polycyclic aromatic amines, fluorescamine-labeled catecholamines, and other fluorescent derivatizing agents such as the coumarins, ninhydrins, o-phthalaldehydes, 7-fluoro-4-nitrobenz-2,1,3-oxadiazoles, naphthalene-2,3-dicarboxaldehydes, cyanobenzf!isoindoles and dansyl chlorides (Simons, S. S., Jr. and D. F. Johnson, J. Am. Chem. Soc. 98:7098-7099 (1976); Roth, M., Anal. Chem. 43:880-882 (1971); Dunges, W., ibid, 49:442-445 (1977); Hill, D. W. et al., ibid, 51:1338-1341 (1979); Lindroth, P. and K. Mopper, ibid, 51:1667-1674 (1979); Sigvardson, K. W. and J. W. Birks, ibid, 55:432-435 (1983); Sigvardson, K. W. et al., ibid, 56:1096-1102 (1984); de Montigny, P. et al., ibid, 59:1096-1101 (1987); Grayeski, M. L. and J. K. DeVasto, ibid, 59:1203-1206 (1987); Rubinstein, M. et al., Anal. Biochem. 95:117-121 (1979); Kobayashi, S.-I., et al., ibid, 112:99-104 (1981); Watanabe, Y. and K. Imai, ibid, 116:471-472 (1981); Tsuchiya, H., J. Chromatog. 231:247-254 (1982); DeJong, C. et al., ibid, 241:345-359 (1982); Miyaguchi, K. et al., ibid, 303:173-176 (1984); Sigvardson, K. W. and J. W. Birks, ibid, 316:507-518 (1984); Benson, J. R. and P. E. Hare, Proc. Nat. Acad. Sci. 72:619-622 (1975); Kawasaki, T. et al., Biomed. Chromatog. 4:113-118 (1990)).
There are currently four known nonenzymatic systems: the acridinium derivatives (McCapra et al., British Patent No. 1,461,877; Wolf-Rogers J. et al., J. Immunol. Methods 133:191-198 (1990)); isoluminols, metalloporphyrins (Forgione et al., U.S. Pat. No. 4,375,972) and nonmetallic tetrapyrroles (Katsilometes, PCT International Publication No. WO 93/23756). These systems have certain advantages over the enzyme-mediated systems in that they have faster kinetics resulting in peak light output within seconds. The metalloporphyrins are small hapten molecules which decrease stearic hinderance problems in antigen binding. In addition, the metalloporphyrin molecules known to be luminescent are those containing a paramagnetic metal ion with emission yields above 10.sup.-4 (Gouterman, M., The Porphyrins, Vol. III, Dolphin, D., ed., (Academic Press): 48-50, 78-87, 115-117, 154-155 (1978); Canters, G. W and J. H. Van Der Waals, ibid, 577-578). It has also been known that metalloporphyrins, hyposporphyrins, pseudonormal metalloporphyrins and metalloporphyrin-like molecules such as metallic chlorins, hemes, cytochromes, chlorophylls, lanthanides and actinides undergo oxidation/reduction reactions which are either primary or secondary to structural perturbations occurring in the metallic center of these molecules and that their reactive ability to catalyze the production of chemiluminescence has been ascribed to the metallo center of these molecules (Eastwood, D. and M. Gouterman, J. Mol. Spectros. 35:359-375 (1970); Fleischer, E. B. and M. Krishnamurthy, Annals N.Y. Academy of Sci. 206:32-47 (1973); Dolphin, D. et al., ibid, 206:177-201; Tsutsui, M. and T. S. Srivastava, ibid, 206:404-408; Kadish, K. M. and D. G. Davis, ibid, 206:495-504; Felton, R. H. et al., ibid, 206:504-516; Whitten, D. G. et al., ibid, 206:516-533; Wasser, P. K. W. and J.-H. Fuhrhop, ibid, 206:533-549; Forgione et al., U.S. Pat. No. 4,375,972; Reszka, K. and R. C. Sealy, Photochemistry and Photobiology 39:293-299 (1984); Gonsalves, A. M. d'A. R. et al., Tetrahedron Lett. 32:1355-1358 (1991)). These reactions are altered by iron and other metal ions which may be present in the reactants and these metal ions can interfere with and greatly confound the assay of metalloporphyrin conjugate concentrations (Ewetz, L. and A. Thore, Anal. Biochem. 71:564-570 (1976)). Different metals will strongly influence the lifetimes and luminescent properties of the metalloporphyrins.
The nonmetallic porphyrin deuteroporphyrin-IX HCl has been shown to mediate the production of light from luminol in solution (Katsilometes, G. W., supra).
Use of the luminescent acridinium ester and amide derivatives in chemiluminescent reactions and in the development of nonisotopic ligand binding assays has been reported and reviewed (Weeks, I. et al., Clin. Chem. 29/8:1474-1479 (1983); Weeks, I. and J. S. Woodhead, Trends in Anal. Chem. 7/2:55-58 (1988)). The very short lived emission of photons (&lt;5 sec) to produce the flash-type kinetics in the presence of H.sub.2 O.sub.2 and NaOH oxidation reagents (pH 13.0) is characteristic of the system.
Methods of preparation of acridones and variously substituted acridines and acridones have been summarized (Acridines, Acheson, R. M. and L. E. Orgel, (Interscience Publishers, N.Y.) pp. 8-33, 60-67, 76-95, 105-123, 148-173, 188-199, 224-233 (1956)). Formation of biacridines by the combining of two acridine residues at the carbon-9 atom has been described and reviewed previously (Gleu, K. and R. Schaarschmidt, Berichte 8:909-915 (1940)). These efforts led to the synthesis of 10,10'-dimethyl, 10,10'-diphenyl and 10,10'-diethyl-9,9'-biacridinium nitrate molecules. It was also reported that these molecules will produce light when exposed to hydrogen peroxide in basic solution (Gleu, K. and W. Petsch, Angew. Chem. 48:57-59 (1935); Gleu, K. and R. Schaarschmidt, Berichte 8:909-915 (1940)).
The mechanism of light production by lucigenin (10,10'-dimethyl-9,9'-biacridinium nitrate) has been extensively studied and has been ascribed to a series of hydroxide ion nucleophilic additions to acridinium salts and their reduction products (pinacols), culminated by the oxidation of the main end product N-methylacridone (Janzen, E. G. et al., J. Organic Chem. 35:88-95 (1970); Maeda, K. et al., Bul. Chem. Soc. Japan 50:473-481 (1977); Maskiewicz, R. et al., J. Am. Chem. Soc., 101/18:5347-5354 (1979); Maskiewicz, R. et al. ibid, 101/18:5355-5364 (1979)).
Modifications and derivatizations of 10-methyl acridine at the carbon-9 atom have led to the production of several useful chemiluminescent molecules having varying degrees of stability (Law, S.-J., et al., J. Biolum. Chemilum. 4:88-98 (1989)). These molecules produce a flash of light lasting less than five seconds when exposed to 0.5% w/v hydrogen peroxide in 0.1 mol/L nitric acid followed by a separate solution containing 0.25 mol/L sodium hydroxide.
A luminescent derivative, a luminescent derivatized molecule or a derivatized luminescent molecule as defined herein is a molecule which results from the covalent binding of a functional group or a group which changes the chemical reactivity and properties of a precursor molecule leading to the formation of a luminescent molecule suitable for conjugation to an analyte or a particular binding partner one wishes to use in assay development. A N-hydroxy succinimide derivatization of biacridines at one or both of the two 10,10' positions are the preferred luminescent derivatives of the invention. A compound or a molecule is a "derivative" of a first compound or first molecule if the derivative compound or molecule is formed (or can be formed) by reaction of the first compound or first molecule to form a new compound or new molecule either smaller or larger than the first compound or first molecule while retaining at least part of the structure of the first compound or first molecule. As used herein the term "derivative" can also include a "luminescent derivative".
Prior to this invention, synthesis of derivatized luminescent 10,10'-substituted-9,9'-biacridines has not been achieved. The previously known luminescent biacridines (e.g., lucigenin) contain no reactive group(s) which will permit the conjugation of the molecule to another. Until now, the biacridines have been of academic interest only and have been used to study the mechanism of light production and the interactions of reactive ionic species.
The use of luminescent reactions at the surface of light conductive materials (e.g., fiber-optic bundle) is the basis of the development of luminescent sensors or probes (Blum, L. J. et al., Anal. Lett. 21:717-726 (1988)). This luminescence may be modulated by specific protein binding (antibody) and can be produced in a microenvironment at the surface of the probe. The light output is then measured by photon measuring devices in the formulation of homogeneous (separation free) assays (Messeri, G. et al., Clin. Chem. 30:653-657 (1984); Sutherland, R. M. et al., Complementary Immunoassays, Collins, W. P., ed., (John Wiley & Sons, Ltd.) pp. 241-261 (1988)).
It has been demonstrated that charged synthetic polymers (poly-N-ethyl-4-vinylpyridinium bromide, PEVP) can completely inhibit the production of light by charged conjugate molecules through electrostatic interactions. This has particularly been studied in the enhanced luminol chemiluminescent reaction catalyzed by the negatively charged peroxidase enzyme. Addition of low-molecular-weight electrolytes will eliminate this inhibition thereby supporting an electrostatic nature of the observed effect (Valsenko, S. B. et al., J. Biolum. Chemilum. 4:164-176 (1989)).
Luminescent capillary electrophoresis gels, gel transfers or blots (Southern, Western, Northern and Dot) are examples of techniques which provide quantitative measurement of proteins and nucleic acid genetic material. These techniques can be used in conjunction with methods which amplify analyte expression, e.g., probes, PCR (polymerase chain reaction) bands, RFLP (restriction fragment length polymorphisms) methods and other methods which amplify gene expression and other analytes (Stevenson, R., Biotech. Lab. 8:4-6 (1990)).
It would be beneficial in improving assay sensitivity to increase the output of light obtained from chemiluminescent reactions by synthesizing biacridine molecules capable of producing superior quantities of photo emissions and by improving existing signal solutions and to have novel signal solutions which provide a greater intensity of light during chemiluminescent reactions. The ability to modulate the kinetics of light output through manipulation of the signal solution formula is particularly beneficial in tailoring assays for a variety of uses (genetic probe, sensor, hormones, etc.).